Not applicable.
This invention is directed to methods for producing combinatorial chemistry libraries containing mercapto (thiol) compounds and derivatives. This invention is further directed to synthesis of combinatorial chemistry libraries of mercapto compounds and derivatives using solid-phase techniques. This invention is still further directed to the libraries of mercapto compounds and derivatives produced by the synthetic methods disclosed. This invention is still further directed to utilizing the libraries of mercapto compounds and derivatives thereof to identify and select compounds which bind to, inhibit, or otherwise affect enzymes, receptors, or other biological molecules implicated in disease processes (including disease-related metalloproteinases). The mercapto compounds and derivatives thus selected are suitable for use as therapeutics.
The techniques of combinatorial chemistry have been increasingly exploited in the process of drug discovery. Combinatorial chemistry allows for the synthesis of a wide range of compounds with varied molecular characteristics. Combinatorial synthetic techniques enable the synthesis of hundreds to millions of distinct chemical compounds in the same amount of time required to synthesize one or a few compounds by classical synthetic methods. Subjecting these compounds to high-throughput screening allows thousands of compounds to be rapidly tested for desired activity, again saving time, expense and effort in the laboratory.
Chemical combinatorial libraries are diverse collections of molecular compounds. Gordon et al. (1995) Acc. Chem. Res. 29:144-154. These compounds are formed using a multistep synthetic route, wherein a series of different chemical modules can be inserted at any particular step in the route. By performing the synthetic route multiple times in parallel, each possible permutation of the chemical modules can be constructed. The result is the rapid synthesis of hundreds, thousands, or even millions of different structures within a chemical class.
For several reasons, the initial work in combinatorial library construction focused on peptide synthesis. Furka et al. (1991) Int. J. Peptide Protein Res. 37:487-493; Houghten et al. (1985) Proc. Natl. Acad. Sci. USA 82:5131-5135; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81: 3998-4002; and Fodor et al. (1991) Science 251:767. The rapid synthesis of discrete chemical entities is enhanced where the need to purify synthetic intermediates is minimized or eliminated; synthesis on a solid support serves this function. Construction of peptides on a solid support is well-known and well-documented. Obtaining a large number of structurally diverse molecules through combinatorial synthesis is furthered where many different chemical modules are readily available; hundreds of natural and non-natural amino acid modules are commercially available. Finally, many peptides are biologically active, making them suitable for use as a class to the pharmaceutical industry.
The scope of combinatorial chemistry libraries has recently been expanded beyond peptide synthesis. Polycarbamate and N-substituted glycine libraries have been synthesized in an attempt to produce libraries containing chemical entities that are similar to peptides in structure, but possess enhanced proteolytic stability, absorption and pharmacokinetic properties. Cho et al. (1993) Science 261:1303-1305; and Simon et al. (1992) Proc. Natl. Acad. Sci. USA 89:9367-9371. Furthermore, benzodiazepine, pyrrolidine, and diketopiperazine libraries have been synthesized, expanding combinatorial chemistry to include heterocyclic entities. Bunin et al. (1992) J. Am. Chem. Soc. 114:10997-10998; Murphy et al. (1995) J. Am. Chem. Soc. 117:7029-7030; and Gordon et al. (1995) Bioorg. Medicinal Chem. Lett. 5:47-50.
Mercapto compounds have received a great deal of attention as inhibitors of matrix metalloproteinases (MMPs), a class of enzymes that acts on components of the extracellular matrix and basement membranes, such as collagen, fibronectin, and laminin. Borden et al. (1997) Critical Reviews in Eukaryotic Gene Expression 7:159. The extracellular matrix plays a role in a wide variety of biological processes, such as cell proliferation, cell differentiation, cell adhesion, cell migration, and tissue morphogenesis. Matrisian (1990) Trends in Genetics 6:121. Metalloproteinases are members of a superfamily of enzymes which share a number of features. Their activity depends on the peptide nature of their substrates; full enzymatic activity requires a metal ion (generally zinc, cobalt, or iron) bound by the side chains of conserved amino acids at or near the active site; among the conserved metal-binding residues are histidines belonging to a motif, HEXXH (SEQ ID NO:1). The enzymes are sensitive to metal-chelating reagents. Vallee and Auld (1990) Biochem. 29:5647-5659; and Stxc3x6cker et al. (1995) Protein Sci. 4: 823-840. Currently, the family comprises two subclasses, exemplified by thermolysin and metzincins.
The metalloproteinase superfamily encompasses metalloproteinases from a wide variety of organisms. For example, matrix metalloproteinases in mammals act to modify or degrade extracellular matrix components such as collagens, fibronectin, and laminin. Birkedal-Hansen et al. (1993) Crit. Rev. Oral Biol. Med. 4:197-250. MMP""s are believed to be involved in the development of arthritis, tumor angiogenesis, retinopathy, and many other disease processes. While many MMP""s are secreted from the cell, others remain membrane bound. Takino et al. (1995) J. Biol. Chem. 270:23013-23020; Will and Hinzmann (1995) Eur. J. Biochem. 231:602-608; and Turner and Tanzawa (1997) FASEB J. 11:355-364. Other metalloproteinases isolated from mammals include endopeptidase EC 3.4.24.15, which is believed to be involved in the regulated metabolism of a number of neuropeptides (Papastoitsis et al. (1994) Biochem. 33:192-199; and McDermott et al. (1992) Biochem. Biophys. Res. Comm. 185:746-753); angiotensin-converting enzyme; endothelin-converting enzyme; and neutral endopeptidase. Homologues of these various human metalloproteinases have been reported in a variety of animal species. Snake venom metalloproteinases also degrade major proteins of the extracellular matrix, and further have been reported to degrade platelet integrin VLA-2 and von Willebrand factor. Jia et al. (1996) Toxicon 34:1269-1276; and Kamiguti et al. (1996) Toxicon 34:627-642. Fungi such as Aspergillus and Fusarium have been reported to synthesize metalloproteinases. Sekine (1973) Agric. Biol. Chem. 37:1945-1952; and U.S. Pat. No. 5,691,162. Metalloproteinases have also been isolated from parasitic organisms which can be pathogenic toward mammals, including protozoan parasites such as helminths (U.S. Pat. No. 5,691,186). Bacteria also synthesize metalloproteinases. Hxc3xa4se et al. (1993) Microbiol. Rev. 57:823-837. Metalloproteinases have been isolated from various bacteria including Bacillus species such as Bacillus subtilis (McConn et al. (1964) J. Biol. Chem. 239:3706); Serratia (Miyata et al. (1971) Agr. Biol. Chem. 35:460); Legionella pneumophila (Moffat et al. (1994) Infection and Immunity 62:751-753); Vibrio species (Takahashi et al. (1996) Biosci. Biotech. Biochem. 60:1651-1654; and Clostridium species such as Clostridium perfringens (Minami et al. (1997) Microbiol. Immunol. 41:527-535. Activities of some of these enzymes can produce deleterious effects in mammals. For example, the xcex-toxin of C. perfringens acts to cleave and activate another toxin produced by this bacterium. Minami et al. (1997). Other bacterial metalloproteinases can act to activate zymogen forms of human MMP""s. Okamoto et al. (1997) J. Biol. Chem. 272:6059-6066.
A relatively new member of the metalloproteinase superfamily is the bacterial enzyme peptide deformylase (PDF). In bacteria, nascent proteins typically contain an N-formyl group on the N-terminal methionine. This enzyme catalyzes removal of the formyl moiety from nascent proteins, and this activity is essential for maturation of nascent proteins. Deformylase activity is critical to the growth of Escherichia coli. Chang et al. (1989) J. Bacteriol. 171:4071-4072; and Meinnel and Blanquet (1994) J. Bacteriol. 176:7387-7390. While this enzyme clearly shares many of the features which characterize metalloproteinases, it differs from other members of the superfamily in several important respects. Firstly, the metal ion in the active enzyme appears to be Fe(II), or possibly another divalent cationic metal, instead of the zinc ion more commonly encountered. Rajagopalan et al., (1997) J. Am. Chem. Soc., 119:12418-19. Secondly, the divalent ion appears to play an important role, not only in catalysis, but also in the structural integrity of the protein; thirdly, the third ligand of the divalent ion is a cysteine, rather than a histidine or a glutamate, as in other metalloproteinases; fourthly, this third ligand is not located at the C-terminal side of the HEXXH (SEQ ID NO:1) motif but far away along the amino acid sequence, and N-terminal to the motif; finally, the solution structure shows significant differences in the secondary and tertiary structure of PDF, compared with other prototypical metalloproteinases. Meinnel et al. (1996) J. Mol. Biol. 262:375-386. PDF from E. coli, Bacillus stearothermophilus, and Thermus thermophilus have been characterized. Meinnel et al. (1997) J. Mol. Biol. 267:749-761. The enzyme studied by Meinnel et al. contained a zinc ion as the divalent ion, and the structural features summarized above were obtained from zinc-containing proteins.
MMPs present an attractive target for therapy in a wide variety of disorders due to the role they play in both normal and pathological physiological processes. These pathologies include tumor growth, invasion and metastasis (Coussens et al. (1996) Chemistry and Biology 3:895; Brown et al. (1995) Ann. Oncol. 6:967; and McDonnell et al. (1990) Cancer and Metastasis Review 9:305); angiogenesis during cancer development (Liotta et al. (1991) Cell 64:327); abdominal aortic aneurysms (Thompson, (1996) Curr. Opin. Cardiol. 11:504); inflammation (Goetzl et al. (1996) J. Immunol. 156:1); and arthritis (Murphy et al. (1992) J. Rheumatol. (suppl. 32) 19:61). Gelatinase A (Type IV collagenase) has been implicated in breaching the blood-brain barrier during hemorrhagic brain injury (Rosenberg et al. (1995) Brain Res. 703:151). Other potential enzyme targets of clinical interest include microbial enzymes (Onishi et al.(1996) Science 274:980).
Because of the wide variety of potential clinical applications for MMP inhibitors, much attention has been focused on developing such inhibitors. Brown (1995) Advan. Enzyme Regul. 35:293. Thiol-based collagenase inhibitors are reported in Johnson et al. (1987) J. Enzyme Inhibition 2:1. Mercapto inhibitors of collagenase and stromelysin inhibitors are described in Wahl et al. Chapter 19 of Annual Reports in Medicinal Chemistry, Vol. 25, Academic Press, San Diego, 1989. Thiol inhibitors of proteoglycanases and collagenases are disclosed in DiPasquale et al. (1986) Proc. Soc. Exp. Biol. Med. 183:262. Thiol and other inhibitors of collagenases are described in Schwartz et al. Chapter 8 in Progress in Medicinal Chemistry, vol. 29, Elsevier, N.Y., 1992.
With one important exception, efforts in the field of combinatorial chemistry have not focused on mercapto compounds (thiol-containing compounds). This exception is, of course, peptides containing cysteine residues and analogs such as homocysteine. The free side chain of cysteine contains a mercapto (thiol) group, and hence a cysteine-containing peptide can be considered a mercapto compound. If compounds which have thiol groups provided solely by one or more cysteine, homocysteine, or other cysteine analogs are excluded from the purview of mercapto libraries, however, the field is substantially narrowed.
One previous effort in generating combinatorial libraries of mercapto compounds on a solid support is presented in Murphy et al. (1995) J. Am. Chem. Soc. 117:7029-7030, and in the related work presented in U.S. Pat. Nos. 5,525,734 and 5,525,735 and in International Patent Application WO 95/35278. All of these references disclose a method of synthesizing a combinatorial library of pyrrolidine compounds. Incorporation of the mercapto moiety is accomplished by coupling a mercapto acyl halide to the pyrrolidine nitrogen of the support-bound compounds.
A method of rapidly generating MMP inhibitors would find immediate application in generating lead compounds for pharmaceutical investigation. This invention provides methods for rapidly generating thiol compounds, which can then be subjected to high-throughput pharmacological screening for identification of compounds for further study.
All references, publications and patents mentioned herein are hereby incorporated by reference herein in their entirety.
The present invention provides methods for synthesizing combinatorial libraries of mercapto compounds and their derivatives on a solid support. The invention also encompasses the combinatorial libraries prepared by the synthetic methods of the invention. One method for synthesizing the mercapto compounds comprises adding one or more nucleophiles containing an S-protected mercapto group to a solid support wherein the solid support comprises a leaving group and an insoluble portion. The nucleophile displaces the leaving group on the solid support, producing an S-protected mercapto compound(s) bound to the insoluble portion. The solid support-bound mercapto compound(s) are derivatized by one or more chemical transformations, followed by deprotection of the mercapto group and cleavage of the compounds from the solid support. The deprotection of the mercapto group can be performed either before or after the cleavage of the compounds from the solid support.
In another method, a compound or compounds comprising a free mercapto group react with a solid support comprising a leaving group. The mercapto group replaces the leaving group on the solid support. The compound or compounds are then further derivatized, followed by cleavage of the compounds from the solid support.
In one embodiment, the methods are used to synthesize compounds and libraries of compounds of the formulas: 
wherein each of R1, R2, R3, R4, R5, R6, R7, and R8 are independently selected from the group consisting of H, alkyl, heteroalkyl, aryl, heteroaryl, and heterocyclic moieties as defined herein, as well as amino acid side chains (both naturally and non-naturally occurring as defined herein), and where n is an integer from 1 to 5, m is an integer from 0 to 5, p is an integer from 1 to 7, and a and b are integers ranging independently from 1 to 8 with the proviso that (a+b)xe2x89xa69. The R groups can be attached to asymmetric carbon atoms in either the R-configuration or the S-configuration; additionally, all stereoisomeric and diastereomeric variations of the compounds and substituents are included in the invention. All protected derivatives of the compounds and all salts of the compounds are also included in the invention.
The methods of the invention also enable synthesis of compounds represented by the structure 
where one of R50, R51, R52, R53, and R54 is xe2x80x94N(R55)(R56), xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94R55, or xe2x80x94C(xe2x95x90O)xe2x80x94N(R55)(R56), and the remainder of R50, R51, R52, R53, and R54, as well as R55 and R56, are independently selected from the group consisting of H, alkyl, heteroalkyl, aryl, heteroaryl, and heterocyclic moieties as defined herein, as well as amino acid side chains (both naturally and non-naturally occurring as defined herein). All stereoisomeric and diastereomeric variations of the compounds and substituents are included in the invention. All protected derivatives of the compounds and all salts of the compounds are also included in the invention.
In another embodiment, the invention encompasses methods of synthesizing a combinatorial library of mercapto compounds. In this embodiment, the nucleophile(s) is selected from the group consisting of compounds having the formula 
wherein R1, R2, R3, R4, R5, and R6 are independently selected from the group consisting of H, alkyl, heteroalkyl, aryl, heteroaryl, and heterocyclic moieties, and amino acid side chains; and where n is an integer from 1 to 5; p is an integer from 1 to 7; and a and b are independently integers from 1 to 8, with the proviso that (a+b)xe2x89xa69.
In another embodiment, the invention encompasses further methods of synthesis of a combinatorial library of mercapto compounds. In this embodiment, the nucleophile(s) is selected from the group consisting of compounds having the formula 
where one of R50, R51, R52, R53, and R54 is xe2x80x94NH2, xe2x80x94COOH, or xe2x80x94C(xe2x95x90O)xe2x80x94NH2, and the remainder of R50, R51, R52, R53, and R54 are independently selected from the group consisting of H, alkyl, heteroalkyl, aryl, heteroaryl, and heterocyclic moieties and amino acid side chains.
In another embodiment, the invention also encompasses methods of synthesizing combinatorial libraries of mercapto compounds, where the nucleophile is S-trityl-2-aminoethanethiol.
In another embodiment, the invention also encompasses methods of synthesizing combinatorial libraries of mercapto compounds, where a mercaptoamine is added to a resin, with the mercapto group bound to the resin and the amino group is available for further reaction. In another embodiment, the resin is trityl alcohol resin and the mercaptoamine is 2-aminoethanethiol.
The invention also encompasses the combinatorial library or libraries of the compounds synthesized by the combinatorial method described above. These libraries are composed of a plurality of compounds from one or more classes of the compounds described above (formulas I-XVII and XVIII). Depending on the procedure used for synthesis, the combinatorial library preferably contains at least about 80, 160, 320, 640, 1000, 5000, 10,000, 50,000, 100,000, 500,000, 1,000,000, or more than 1,000,000 distinct compounds.
The invention also encompasses an S-protected mercapto compound functionalized resin, prepared by adding one or more nucleophile(s) comprising an S-protected mercapto group and a free nucleophilic amino group to a solid support.
The invention also encompasses linkers suitable for attachment to an amine-bearing resin, comprising an acid-labile linker group and an S-protected mercapto group.
The invention also encompasses the compounds represented by the formulas: 
where b is an integer from 1 to 5 and P1 is a protecting group selected from the group consisting of trityl, p-methoxytrityl, p-methyltrityl, acetamidomethyl, benzyl, t-butyl, t-butylthio, and p-methoxybenzyl protecting groups.
The invention also encompasses the compounds represented by the formulas: 
where b is an integer from 1 to 5 and P1 is a protecting group selected from the group consisting of trityl, p-methoxytrityl, p-methyltrityl, acetamidomethyl, benzyl, t-butyl, t-butylthio, and p-methoxybenzyl protecting groups.
The invention also encompasses the compounds represented by the formulas: 
where b is an integer from 1 to 5; and where b is 3.
The invention also encompasses the compounds represented by the formulas: 
where b is an integer from 1 to 5, and where b is 3.
The invention also encompasses the compounds represented by the formula 
where b is an integer from 1 to 5, P1 is a protecting group selected from the group consisting of trityl, p-methoxytrityl, p-methyltrityl, acetamidomethyl, benzyl, t-butyl, t-butylthio, and p-methoxybenzyl protecting groups, and RESIN is any solid or polymeric support.
The invention also encompasses the compounds represented by the formula 
where b is an integer from 1 to 5, and RESIN is any solid or polymeric support.
The invention also encompasses the compounds represented by the formula 
where b is an integer from 1 to 5, P1 is a protecting group selected from the group consisting of trityl, p-methoxytrityl, p-methyltrityl, acetamidomethyl, benzyl, t-butyl, t-butylthio, and p-methoxybenzyl protecting groups, and RESIN is any solid or polymeric support.
The invention also encompasses the compounds represented by the formula 
where b is an integer from 1 to 5, and RESIN is any solid or polymeric support.
The invention also encompasses the aforementioned compounds where RESIN-NHxe2x80x94 is Tentagel S NH2 resin in an amide linkage to the remainder of the compound, and/or where b is 3.
The invention also encompasses methods of use of the libraries obtained by the combinatorial methods. The uses include screening for bioactive compounds, by contacting the library with an enzyme, receptor, or cell under conditions conducive to specific binding, and isolating the mercapto compound or compounds that specifically bind to the enzyme, cell, or receptor.
The invention also encompasses methods of use of the libraries synthesized by the combinatorial methods to screen for pharmacologically active compounds. In one embodiment, the invention provides for a method for screening for inhibitors of the enzyme deformylase, by contacting the deformylase with a mercapto compound and determining the inhibition of the enzyme by the compound. In another embodiment, the invention provides a method for determining the antimicrobial efficacy of a compound, by screening compounds for their ability to inhibit deformylase, and then determining the antimicrobial activity of inhibitors of deformylase. In still another embodiment, the invention provides for compositions of matter which have antimicrobial activity or which inhibit a deformylase enzyme, and methods for inhibiting deformylase, affecting microbial growth, and treating microbial infections by administering the compositions of matter.
The invention also encompasses the bioactive compounds discovered by contacting the library with an enzyme, receptor, or cell under conditions conducive to specific binding, and isolating the mercapto compound or compounds that specifically bind to the enzyme, cell, or receptor. The invention also encompasses the bioactive compounds in combination with a pharmaceutically acceptable carrier.
The invention also encompasses use of the bioactive compounds in combination with a pharmaceutically acceptable carrier in treating diseases in mammals. Suitable diseases include, but are not limited to tumor growth, tumor invasion, tumor metastasis, angiogenesis during cancer development, abdominal aortic aneurysms, inflammation, arthritis, and hemorrhagic brain injury.